Infection represents one of the most fundamental threats to host integrity. When bacterial pathogens breach an epithelial barrier, the host innate immune system detects the attack and triggers a response to contain and eliminate the invader. Cellular sensors, such as macrophages and dendritic cells, detect pathogen through receptors that recognize bacterial macromolecules. These cells then mount a proinflammatory reaction that leads to cellular responses that contain and eliminate the infection. Thus, the innate immune response contains both afferent (pathogen-sensing) and efferent (proinflammatory) limbs. The Toll/IL-1 signal transduction pathway mediates both arms of this innate response to infection. This conserved signaling cascade consists of the proteins MyD88, the interleukin-1 receptor-associated kinase (IRAK) family of molecules (IRAK, IRAK2, and IRAK-M) and the tumor necrosis factor associated factor 6. It processes signals from at least 10 Toll-like receptors (TLRs) and three IL-1 receptor family members (IL-1, IL-18, and Ti/ST2), distributing them to multiple downstream targets, including NF-kB and several mitogen activated protein kinase cascades. We have genetically deleted IRAK, the primary proximal kinase in this pathway, in mice. IRAK-deficient animals and macrophages exhibit impaired responses to lipopolysachharide (LPS), peptidoglycan (PGN), lipotechoic acid (LTA), and CpG DNA, bacterial molecules that activate the afferent arm of innate immunity through TLR4 and TLR2. These mice also exhibit attenuated proinflammatory (efferent) responses due to disrupted IL-1 and IL-18 signaling. The overall objective of this proposal is to determine IRAK function in the host response to Gram-negative and Gram-positive infections. Aim 1 is to determine the role of IRAK in the acute inflammatory response to Gram-negative and Gram-positive infections. We will subject IRAK- deficient macrophages and mice to increasingly complex models of these infections, using toxin challenges, stimulation with nonreplicating bacteria, and Klesiella pneumoniae and Staphylococcus aureus pneumonia. Aim 2 is to isolate IRAK function genetically to either the TLR4 or IL-1receptor pathway by generating IRAK/IL-121 and IRAK/TLR4 double knockout animals and comparing the responses of double and single KO macrophages and mice to LPS stimulation, nonreplicating K. pneumoniae, and K. pneumoniae pneumonia. Aim 3 is to determine the contributions of IRAK2 and IRAK-M to residual TLR signaling in IRAK-deficient cells, as deletion of IRAK impairs, but does not completely abrogate signaling. These studies should provide fundamental new information about the role of IRAK in the innate immune response to acute bacterial infection and may eventually lead to the development of strategies to modulate deleterious aspects of this response.